LOX-mediated ECM mechanical stress induces Piezo1 activation in hypoxic-ischemic brain damage and identification of novel inhibitor of LOX.

Hypoxic-ischemic encephalopathy (HIE) poses a significant challenge in neonatal medicine, often resulting in profound and lasting neurological deficits. Current therapeutic strategies for hypoxia-ischemia brain damage (HIBD) remain limited. Ferroptosis has been reported to play a crucial role in HIE and serves as a potential therapeutic target. However, the mechanisms underlying ferroptosis in HIBD remain largely unclear. In this study, we found that elevated lysyl oxidase (LOX) expression correlates closely with the severity of HIE, suggesting LOX as a potential biomarker for HIE. LOX expression levels and enzymatic activity were significantly increased in HI-induced neuronal models both in vitro and in vivo. Notably, we discovered that HI-induced brain tissue injury results in increased stiffness and observed a selective upregulation of the mechanosensitive ion channel Piezo1 in both brain tissue of HIBD and primary cortex neurons. Mechanistically, LOX increases its catalytic substrates, the Collagen I/III components, promoting extracellular matrix (ECM) remodeling and possibly mediating ECM cross-linking, which leads to increased stiffness at the site of injury and subsequent activation of the Piezo1 channel. Piezo1 senses these stiffness stimuli and then induces neuronal ferroptosis in a GPX4-dependent manner. Pharmacological inhibition of LOX or Piezo1 ameliorated brain neuronal ferroptosis and improved learning and memory impairments. Furthermore, we identified traumatic acid (TA) as a novel LOX inhibitor that effectively suppresses LOX enzymatic activity, mitigating neuronal ferroptosis and promoting synaptic plasticity. In conclusion, our findings elucidate a critical role for LOX-mediated ECM mechanical stress-induced Piezo1 activation in regulating ferroptotic cell death in HIBD. This mechanistic insight provides a basis for developing targeted therapies aimed at ameliorating neurological outcomes in neonates affected by HIBD.

L-arginine loading porous PEEK promotes percutaneous tissue repair through macrophage orchestration.

Infection and poor tissue repair are the key causes of percutaneous implantation failure. However, there is a lack of effective strategies to cope with due to its high requirements of sterilization, soft tissue healing, and osseointegration. In this work, l-arginine (L-Arg) was loaded onto a sulfonated polyetheretherketone (PEEK) surface to solve this issue. Under the infection condition, nitric oxide (NO) and reactive oxygen species (ROS) are produced through catalyzing L-Arg by inducible nitric oxide synthase (iNOS) and thus play a role in bacteria sterilization. Under the tissue repair condition, L-Arg is catalyzed to ornithine by Arginase-1 (Arg-1), which promotes the proliferation and collagen secretion of L929 and rBMSCs. Notably, L-Arg loading samples could polarize macrophages to M1 and M2 in infection and tissue repair conditions, respectively. The results in vivo show that the L-Arg loading samples could enhance infected soft tissue sealing and bone regeneration. In summary, L-Arg loading sulfonated PEEK could polarize macrophage through metabolic reprogramming, providing multi-functions of antibacterial abilities, soft tissue repair, and bone regeneration, which gives a new idea to design percutaneous implantation materials.

Both prokaryotes and eukaryotes produce an immune response against plasmids with 5'-GTTTGTT-3'.

In the evolutionary "arms race" from prokaryotes to eukaryotes, some memories of foreign DNA have been conserved for defensive purposes. Shortly after invasion by the plasmid, pEGFP-N1, the conserved the defense gene, isg15, was activated in the zebrafish zygote and in mammalian cells. Based on the sequence similarity, we found three virus-derived sequences in pEGFP-N1 which share the 5'-GTTTGTT-3' core sequence, an epigenetic factor leading to increased expression of isg15. Mutation of the core sequence greatly reduces the degradation rate of the plasmid in E. coli cells or zebrafish embryos. We conclude that a conserved defense response, common to both eukaryotic and prokaryotic cells, allows identification and degradation of plasmids containing 5'-GTTTGTT-3'.

VPS28 regulates brain vasculature by controlling neuronal VEGF trafficking through extracellular vesicle secretion.

Extracellular vesicles (EVs) participate in intercellular communication and contribute to the angiogenesis. However, the understanding of the mechanisms underlying EVs secretion by neurons and their action on the vascular system of the central nervous system (CNS) remain rudimentary. Here, we show that vacuolar protein sorting 28 (Vps28) is essential for the sprouting of brain central arteries (CtAs) and for the integrity of blood-brain barrier (BBB) in zebrafish. Disruption of neuron-enriched Vps28 significantly decreased EVs secretion by regulating the formation of intracellular multivesicular bodies (MVBs). EVs derived from zebrafish embryos or mouse cortical neurons partially rescued the brain vasculature defect and brain leakage. Further investigations revealed that neuronal EVs containing vascular endothelial growth factor A (VEGF-A) are key regulators in neurovascular communication. Our results indicate that Vps28 acts as an intercellular endosomal regulator mediating the secretion of neuronal EVs, which in turn communicate with endothelial cells to mediate angiogenesis through VEGF-A trafficking.

Zebrafish Foxc1a controls ventricular chamber maturation by directly regulating wwtr1 and nkx2.5 expression.

Chamber maturation is a significant process in cardiac development. Disorders of this crucial process lead to a range of congenital heart defects. Foxc1a is a critical transcription factor reported to regulate the specification of cardiac progenitor cells. However, little is known about the role of Foxc1a in modulating chamber maturation. Previously, we reported that foxc1a-null zebrafish embryos exhibit disrupted heart structures and functions. In this study, we observe that ventricle structure and cardiomyocyte proliferation are abolished during chamber maturation in foxc1a-null zebrafish embryos. To observe the endogenous localization of Foxc1a in the hearts of living embryos, we insert eyfp at the foxc1a genomic locus using TALEN. Analysis of the knockin zebrafish show that foxc1a is widely expressed in ventricular cardiomyocytes during chamber development. Cardiac RNA sequencing analysis reveals the downregulated expression of the Hippo signaling effector wwtr1. Dual-luciferase and chromatin immunoprecipitation assays reveal that Foxc1a can bind directly to three sites in the wwtr1 promoter region. Furthermore, wwtr1 mRNA overexpression is sufficient to reverse the ventricle defects during chamber maturation. Conditional overexpression of nkx2.5 also partially rescues the ventricular defects during chamber development. These findings demonstrate that wwtr1 and nkx2.5 are direct targets of Foxc1a during ventricular chamber maturation.

Attractin Participates in Schizophrenia by Affecting Testosterone Levels.

Attractin (ATRN) is a widely expressed glycoprotein that is involved in energy homeostasis, neurodevelopment, and immune response. It is encoded by a gene spanning 180 kb on chromosome 20p13, a region previously implicated in schizophrenia by linkage studies. To address a possible role of ATRN in disorders of the central nervous system, we created an atrn knockout zebrafish line and performed behavioral tests. Adult atrn-/- zebrafish exhibited more pronounced attack behavior relative to wild-type control zebrafish in a tracking analysis. Biochemical analysis revealed elevated testosterone levels in atrn-/- zebrafish. At the gene expression level, we noted an upregulation of cyp51 and hsd17b7, key proteins in testosterone synthesis in the brains of both adult and larvae of atrn-/- zebrafish. In order to further elucidate the relationship between testosterone and behavioral syndromes, we then compared testosterone levels of 9,008 psychiatric patients and 247 healthy controls from the same catchment area. Of all subjects examined, male subjects with schizophrenia exhibited lower testosterone levels compared with controls. In contrast, female subjects with a diagnosis of schizophrenia or bipolar disorder featured higher testosterone levels than did same sex controls. Purposeful sampling of extreme groups showed reduced ATRN expression in a subset of these subjects. Finally, we identified 14 subjects with ATRN mutations. All of whom displayed abnormal testosterone levels. In summary, the interplay of ATRN and testosterone may help to explain sexual dimorphisms in selected behavioral phenotypes.

Axenfeld-Rieger syndrome-associated mutants of the transcription factor FOXC1 abnormally regulate NKX2-5 in model zebrafish embryos.

FOXC1 is a member of the forkhead family of transcription factors, and whose function is poorly understood. A variety of FOXC1 mutants have been identified in patients diagnosed with the autosomal dominant disease Axenfeld-Rieger syndrome, which is mainly characterized by abnormal development of the eyes, particularly those who also have accompanying congenital heart defects (CHD). However, the role of FOXC1 in CHD, and how these mutations might impact FOXC1 function, remains elusive. Our previous work provided one clue to possible function, demonstrating that zebrafish foxc1a, an orthologue of human FOXC1 essential for heart development, directly regulates the expression of nkx2.5, encoding a transcriptional regulator of cardiac progenitor cells. Abnormal expression of Nkx2-5 leads to CHD in mice and is also associated with CHD patients. Whether this link extends to the human system, however, requires investigation. In this study, we demonstrate that FOXC1 does regulate human NKX2-5 expression in a dose-dependent manner via direct binding to its proximal promoter. A comparison of FOXC1 mutant function in the rat cardiac cell line H9c2 and zebrafish embryos suggested that the zebrafish embryos might serve as a more representative model system than the H9c2 cells. Finally, we noted that three of the Axenfeld-Rieger syndrome FOXC1 mutations tested increased, whereas a fourth repressed the expression of NKX2-5 These results imply that mutant FOXC1s might play etiological roles in CHD by abnormally regulating NKX2-5 in the patients. And zebrafish embryos can serve as a useful in vivo platform for rapidly evaluating disease-causing roles of mutated genes.